In the conduct of certain cell biology research, the growth and maintenance of cells derived from animal and human tissues is essential. For example, tissue culture is used extensively in studies of inherited disorders in both animals and humans. Tissue culture of human muscle cells is of particular interest for the study of human muscle cell disorders. While in vitro growth of human skeletal muscle cells has been achieved, relatively little attention has been given to the development of culture media optimized specifically for these cells.
The development of serum-free media for cell growth allows the identification of cell growth factors and allows detailed biochemical and metabolic studies of the cell. For example, the use of serum-free media for the growth of human muscle cells facilitates the investigation of the role of growth factors in muscle differentiation. Such studies are important in the determination of the biochemical basis of certain muscle disorders.
Clonal growth of mononucleate cells from embryonic chicken skeletal muscle and their fusion and differentiation to form multinucleate contractile myotubes was achieved relatively early in the history of modern cell culture ( see, for example, Konigsberg, I. R. (1963) Science 140:1273-1284). Growth of human skeletal muscle cells in vitro was also achieved quite early (Hauschka, S. D. (1974) Dev. Biol. 37:329-344). However, despite many years of studies on growth and differentiation of skeletal muscle cells in vitro, relatively little attention has been given to the development of culture media optimized specifically for growth of muscle cells from humans or other species.
Both undefined and defined media have been described for the growth of muscle cells in culture. Nutrient medium F10 (Ham, R. G. (1963) Exp. Cell Res. 29:515-526), originally developed for Chinese hamster ovary cells, was used with addition of serum and chicken embryo extract to grow chicken embryo muscle cells (Hauschka, S. D. (1966) Proc. Natl. Acad. Sci. USA 55:119-126) and later for growth of human muscle cells (Hauschka, S. D. (1974) supra). F10 plus serum and embryo extract has been widely used for muscle cell culture, in particular, for the establishment of clonal cultures from normal and diseased human muscle cells (Blau, H. M. and Webster, C. (1981) Proc. Natl. Acad. Sci. USA 78:5623-5627; Blau, H. M. and Webster, C. (1983) Proc. Natl. Acad. Sci. USA 80:4856-4860). The most frequently used alternative is Dulbecco's modified Eagle's medium (DME). Other nutrient media that have been used for muscle cell culture, sometimes in combination with DME, include M199 (Askanas, V. and Engel, W. K. (1975) Neurology 25:58-67; Konigsberg, I. R. (1963) supra), MEM plus nonessential amino acids, pyruvate and additional vitamins (Miranda, A. F. et al. (1979) in Muscle regeneration, Mauro, S. et al. (eds.), Raven Press, New York, pp 453-473), RPMI 1640 (Hayashi, I. and Kobylecki, J. (1982) Cold Spring Harbor Conf. on Cell Prolif. 9:857-865), F12 (Pinset, C. and Whalen, R. G. (1985) Dev. Biol. 108:284-289), F14 (Askanas, V. and Gallez-Hawkins, G. (1985a) Arch. Neurol. 42:749-752; Vogel, Z. et al. (1972) Proc. Natl. Acad. Sci. USA 69:3180-3184), MCDB 104 (Allen, R. E. et al. (1985) In Vitro Cell. Dev. Biol. 21:636-640), and MCDB 201 (Dollenmeier, P. etal. (1981) Exp. Cell Res. 135:47-61). Although a number of these media have become standard in their applications, they are suboptimal for efficient promotion of clonal growth of human muscle satellite cells (HMSC).
Often, the culturing of cell types requires the addition of supplements to a basal nutrient medium. These supplements are generally chemically undefined, for example in the form of serum and embryo extract. For many purposes, the use of an undefined supplement is satisfactory. However, for studying growth, metabolism, and/or differentiation of muscle cells in culture, it is desirable to have a supplement that is defined or semi-defined. The introduction of undefined components to the cell culture can contribute to variability in biochemical study results.
Direct replacement of serum with supplements of better defined composition has generally not been very successful for growth of normal cells in conventional basal nutrient media. The amount of undefined supplementation needed for good growth can be reduced or eliminated by optimizing a particular basal nutrient medium (Ham,R. G. (1984) in Methods for Preparation of Media, Supplements, and Substrata for Serum-Free Animal cell culture, Alan R. Liss, Inc. New York, pp. 3-21). For example, a basal nutrient medium, MCDB 131, optimized for growth of human microvascular endothelial cells supported clonal growth with as little as 0.7% dialyzed fetal bovine serum (dFBS) when also supplemented with epidermal growth factor (EGF) and hydrocortisone (Knedler, A. and Ham, R. G. (1987) In Vitro Cell. and Develop. Biol. 23(7):481-491).
Previous defined media for normal HMSC (Askanas, V. et al. (1985b) Soc. Neurosci. Abstr. 11:936; Delaporte, C. et al. (1986) Biol. Cell. 57:17-22; Yasin, R. and van Beers, G. (1983), in Hormonally Defined Media: A Tool in Cell Biology, Fischer, G. and Weiser, R. (eds.), Springer Verlag, Berlin, pp. 406-410) have utilized either DME or F14 (Vogel, Z. et al. (1972) supra) as the nutrient medium without further optimization. These defined media favor differentiation of HMSC rather than optimal growth. In addition, when these media were used, initial inoculation of dense populations of cells into a serum-containing medium, followed by medium change to the serum-free formulation was required.
Askanas et al. (1985b) supra described a defined medium for the growth of HMSC. Basal nutrient medium F14 was used without further optimization. EGF, insulin, and BSA were included as components of the supplements in the medium.
Delaporte et al. (1986) supra developed three defined media for the growth of HMSC. The basal nutrient medium DME was used without further optimization. Insulin was employed in the supplement used in these media. These media promote differentiation and inhibit myoblast proliferation.
Yasin and van Beers (1983) supra tested two defined media for the growth of HMSC. Again the basal nutrient medium DME was used without further optimization. In one medium, DME was supplemented with 0.5 mg/ml fetuin (Sigma type IV), 10-6 M insulin, and 10-7 M dexamethasone. In the other medium, DME was supplemented with 10-6 M insulin and 0.1 mg/ml L-thyroxine. However, it was found that serum was required for proliferation.
Stemerman et al. (U.S. Pat. No. 4,443,546, issued Apr. 17, 1984) entitled "Process and Composition for Propagating Mammalian Cells," purports to provide a serum-free media for normal mammalian cells. However, the specific media provided were developed for vascular smooth muscle cells and endothelial cells.
In addition to being useful for detailed biochemical and metabolic studies of HMSC, a serum-free medium is useful in producing HMSC relatively free of non-human antigens, which are suitable for transplantation into patients having muscle disorders such as muscular dystrophy. Myoblast transfer therapy has been undertaken in humans to treat Duchenne muscular dystrophy (Law, P. et al. (1990) Adv. Exp. Med. Biol. 280:241-250. In transplantation of HMSC to humans, it is preferred that MHC (major histocompatibility complex) or HLA (human leukocyte antigens) be matched to minimize the necessity of immunosuppressive drugs such as cyclosporin A and FK 506. However, as discussed by Karpati, G. (1990) Adv. Exp. Med. Biol. 208:31-34, immunorejection of cultured myoblasts or satellite cells due to MHC incompatibility may be low because human myoblasts express very little MHC I and essentially no MHC II. Minor histocompatibility factors may also be matched between donor and recipient, though these are difficult to detect in myoblasts.
In addition to minimizing immune rejection arising from major or minor histoincompatibility, immune rejection due to anaphylactic or antibody response to non-human antigens present in the transplanted cells should be avoided. A potential source of nonhuman antigens is the medium supplement which has, prior to the subject application, contained animal serum or extracts. Repeated injections of the cell preparation containing non-human antigens can sensitize the recipient, and perhaps ultimately result in an anaphylactic reaction. Karpati, G., supra, recommends using human serum in the culture medium to reduce this problem. However, since the human serum must be type matched to that of the recipient, the presence of a blood borne disease entity (e.g., HCV) must be considered. If the patient's own serum is used in the medium (typically at 10% (v/v) serum), consideration must be given to the stress on the patient.
Previously, Webster, C. et al. (1988) Exp. Cell Res. 174:252-265, have developed a fluorescence activated cell sorting (FACS) method for sorting of myoblasts from fetal muscle tissue. They have been able to recover 10.sup.4 myoblasts from 0.1 g of fetal tissue. The FACS preparations are reported to be 99% myoblasts. Law, P. et al. (1988) Muscle & Nerve 11:525-533, have used about 10.sup.6 myoblasts in murine muscle transplants. Therefore, the FACS prepared cells must be amplified by culturing if they are to be used for transplantation. During the culturing process, non-human antigens can be introduced by the use of serum-containing supplements in the culture medium.
Moreover, as discussed in Karpati, G., supra, to avoid the presence of other cells such as fibroblasts (which express MHC I), it may be preferable to use clonally grown myoblasts. These also must be amplified in culture to produce cell populations large enough for transplantation.
Thus, there is a clear need for a method of growing human myoblast populations that are relatively free of non-human antigens, and therefore useful in human transplantation. Such populations could be produced by growing myoblasts in serum-free medium.